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Chronic intraventricular infusion with NGF improves LTP in old cognitively-impaired rats
Brain Research 770 Ž1997. 1–9
Research report
Chronic intraventricular infusion with NGF improves LTP in old cognitively-impaired rats Jorge A. Bergado ) , Caridad I. Fernandez, Andres Odalys Gonzalez ´ ´ Gomez-Soria, ´ ´ International Centre for Neurological Restoration, AÕe. 25 a 15805, Cubanacan, ´ Playa 12100, Ciudad de La Habana, Cuba Accepted 30 April 1997
Abstract Aged Ž21 months. cognitively-impaired male Sprague–Dawley rats received intraventricular infusion of nerve growth factor ŽNGF. or cytochrome C ŽCit C. for 14 or 28 days using miniosmotic pumps and were evaluated either 1 week or 3 months after treatment. Groups of untreated young, aged-impaired and aged non-impaired rats were also evaluated. Under narcose recording and stimulating electrodes were stereotactically implanted in the dentate gyrus and the perforant path. The stimulation intensity was individually adjusted to obtain a half-maximal population spike ŽP. for test stimuli and a quarter-maximal for tetanization. The amplitude and latency of P and the slope ŽS. of the field EPSP were determined before and at 2, 5, 15, 30 and 60 min after tetanization at 400 Hz. Paired stimuli at 30 ms interval were also applied before and after tetanization. Aged, cognitively impaired rats showed an absent S potentiation and a delayed P potentiation, both in amplitude and latency, while non-impaired rats behaved like the young controls. Paired pulse inhibition showed no difference among groups before or after tetanization suggesting that the impaired potentiation is not due to an increased retroactive inhibition. NGF treatment ameliorates LTP deficits to levels equivalent to non-impaired rats, while Cit C controls showed no improvement. No differences appear among NGF treated groups, but evidence suggest that the animals evaluated 3 months after treatment developed a stronger potentiation. q 1997 Elsevier Science B.V. Keywords: Long-term potentiation; Aging; Nerve growth factor
1. Introduction Nerve growth factor ŽNGF. is the best characterized member of the neurotrophin family exhibiting actions on the peripheral and central nervous system. Only the population of cholinergic neurons distributed along the basal forebrain nuclei and the cholinergic striatal interneurons respond to NGF within the brain w30x. Basal forebrain cholinergic neurons project to the hippocampus and the cerebral cortex where they presumably receive trophic support from target-derived NGF. A gradual decline in the size and number of this cholinergic population with increasing age have been correlated to the progressive memory decline which accompanies aging, especially its pathological variants like Alzheimer’s disease w6,47x. This hypothesized pathological causality together with the known trophic action of NGF on cholinergic neurons provides a rationale for the experi) Corresponding author. [email protected]
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0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 6 1 0 - 0
mental studies on the possible therapeutic usefulness of a NGF-based therapy for age-related cognitive impairments. The experimental evidence shows, coincidently, that intracerebral infusion of NGF to aged animals leads to an improvement of their mnemonic capabilities w22,28x which has been attributed to the enhanced cholinergic function in the targets, specially the hippocampus. Since its discovery in the dentate gyrus ŽDG., hippocampal long-term potentiation ŽLTP. has been proposed as a correlate of long-term memory w11x. This assumption is based mainly on analogies because no conclusive evidence linking LTP and memory has yet been presented, but beyond this unsolved issue, most theories of learning consider that Hebbian plastic modifications of synaptic connectivity occur during the formation of memory engrams. LTP is a long-lasting form of synaptic plasticity and appears, therefore, as a suitable model to study the consequences of NGF treatment on synaptic plasticity. Furthermore, LTP have been found to be altered in aged rats in direct relationship to senescent memory dysfunction w5,17x. The question emerges of whether NGF treatment
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can alleviate the deficient LTP exhibited by aged, cognitively deficient rats. The present paper constitutes the first approach to this fundamental aspect. Our results indicate that chronic intraventricular infusion of NGF to old-impaired rats is able to improve LTP, adding support to the proposed therapeutic use of NGF for the treatment of age-associated memory dysfunctions and offering new perspectives to the interpretation of behavioural results.
2. Materials and methods 2.1. Animals Ten young Ž2 months. and 66 old Ž21 months at the beginning of the experiment. Sprague–Dawley male rats obtained from CENPALAB ŽHavana. were used. The rats were maintained in plastic translucent cages Ž5 animals per cage. under controlled conditions of humidity and temperature with 12:12 h light–dark cycle and free access to food and water during the whole experiment. 2.2. BehaÕioural test Cognitive capacities were evaluated using the Morris Water Maze ŽMWM.. A circular pool with a hidden platform was used. Rats were entered the pool using a pseudorandom distribution of the starting points and the time they need to find the platform was measured. A maximal time of 60 s was allowed in each trial. If after that time the rat was unable to find the escape platform, they were gently carried upon the platform and allowed to stay there for 30 s before the next trial. Eight sessions were performed daily during 5 consecutive days. According to their results, compared with those of a group of ten young rats, old animals were classified as cognitively impaired when their mean latency to find the platform was at least 3
standard deviations above the mean latency of the young animals. The cognitively impaired rats were then randomly assigned to one of the experimental groups. From the non impaired rats some animals were selected at random to constitute one experimental group Žsee Table 1.. The rest of the non impaired rats were discarded an not considered part of the experimental protocol. 2.3. NGF treatment Groups of old-cognitively impaired rats received either 5S murine NGF obtained from mouse submaxillary gland, or Cytochrome C ŽCit C. protein treatment using 14 days continuous delivery miniosmotic pumps ŽAlzet, CA.. Pumps were filled with one of the proteins dissolved in saline and primed before being intrascapullary implanted under ketamine anaesthesia. The pumps were connected through a polyethylene catheter to a stainless steel cannula implanted into the right ventricle. Those animals receiving the substance for 28 days were reanesthetised two weeks after the initial implant and the empty pump was changed by a full one containing the same substance. The concentration of either protein in the pump was 22 m grml, equivalent to an infusion of 1.2 m grday. 2.4. Grouping According to the treatment received the rats were divided in different groups Žsee Table 1.. A control group of young rats, a group of aged non-cognitively impaired rats, and a group of aged animals with cognitive impairment received no treatment before the electrophysiological study. Meanwhile, several subgroups of aged-impaired rats received NGF or Cit C treatment for 14 or 28 days of continuous infusion and were evaluated 1 week or 3 months after the end of substance delivery. As no statistical difference was found among the Cit C treated animals when compared to each other, they were considered as one
Table 1 Experimental groups Group
n
Animals
Substance
Infusion time
Delay
Control Aged non-impaired Aged impaired C 14–1 C 14–3 C 28–1 C 28–3 Cit C N 14–1 N 14–3 N 28–1 N 28–3 NGF
10 9 7 5 5 8 7 25 5 8 6 6 25
Young rats Old rats without cognitive impairment Old rats cognitively impaired Old impaired rats Old impaired rats Old impaired rats Old impaired rats Old impaired rats Old impaired rats Old impaired rats Old impaired rats Old impaired rats Old impaired rats
– – – Cit C Cit C Cit C Cit C Cit C NGF NGF NGF NGF NGF
– – – 14 days 14 days 28 days 28 days
– – – 1 week 3 months 1 week 3 months
14 days 14 days 28 days 28 days
1 week 3 months 1 week 3 months
As no difference was found among the Cit C treated groups in any of the measured variables they were further considered as a single Cit C group Ž n s 25.. The NGF-receiving rats were also grouped in this way for the initial presentations of results Ž n s 25..
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single group ŽCit C.. For the sake of clarity in the presentation of results all animals receiving NGF were also presented as one group ŽNGF. at some instances. 2.5. Electrophysiology For the electrophysiological analysis the rats were anaesthetised using an initial bolus of ketamine clorhydrate Ž3.6 mgrkg. which provides a narcose lasting about 45–60 min, suplemented with chloral hydrate Ž140 mgrkg. every 60 min to maintain narcose during the recording time. The rats were mounted in a stereotaxic frame ŽDavid Kopf, USA.. One monopolar recording electrode ŽRNE 300, Rhodes Med. Inst.. and a bipolar stimulating electrode ŽNE 200, Rhodes Med. Inst.. were lowered at the coordinates; AP: y3.8, L: 2.0, V: 4.0 mm. from bregma aiming the hilus of the DG for recording, and AP: y7.5, L: 4.0, V: 4.5 mm. also from bregma, in the medial portion of the perforant path ŽPP. for stimulation. The vertical position of both electrodes was adjusted under inspection of the evoked potentials to obtain the best sensitivity. Two miniscrews were placed over both frontal bones to serve as earth and indifferent electrode respectively. Constant current square single pulses Ž0.1 ms. were delivered to the PP using a SEN-3301 electronic stimulator ŽNihon Kohden, Japan. connected to the animal through a SS-104J isolator ŽNihon Kohden, Japan.. The evoked potentials were registered using a VC-11 memory osciloscope ŽNihon Kohden, Japan. filtered between 1.5 Hz–3 KHz. Four consecutive responses at 0.2 Hz were averaged with a QC-111J analyser ŽNihon Kohden, Japan. and measured on-line. Three variables were determined on each recorded evoked potential Žsee Fig. 1. The slope ŽS. of field excitatory postsynaptic potential Žf-EPSP. was calculated as DVrDt on the first positive deflection of the potential. The amplitude of the population spike ŽP. and its latency from the beginning of the stimulus artifact ŽP-latency. were also measured. An inputroutput curve was constructed for each animal using the P amplitude as measure of the output. On this curve the stimulation intensity needed to evoke a half maximal P was determined and used for every test record-
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ing. For the induction of LTP an intensity evoking a quarter maximal P was utilized. Three control records with 5 min interval were made before the induction of LTP and the values of S, P amplitude and P latency measured and averaged to obtain the basal values of the three variables. Paired pulse stimulation Ž30 ms interstimulus interval. was also performed before the induction of LTP. The degree of inhibition or facilitation was determined using the amplitude ratio ŽP2rP1 . in both potentials expressed as percent. Paired pulse stimulation was also carried out after the last test recording. LTP was induced using 10 trains of 10 impulses each Ž0.1 ms square pulses at 400 Hz. with 10 s intertrain interval. The intensity was adjusted as described above. Test recordings were performed 2, 5, 15, 30 and 60 min after LTP induction. S, P amplitude, and P latency were determined in each record and expressed as percent the respective basal value. 2.6. Statistics Comparisons were performed using the non-parametric analysis of variance of Kruskall–Wallis followed by a Mann–Whitney’s U-test to asses differences between pairs of groups. In every case a p value inferior to 0.05 was considered significant.
3. Results Though all groups of animals were processed in parallel, the results of the non-treated animals will be described first for the sake of clarity. 3.1. Basal Õalues The basal P amplitude measured before the induction of LTP was significantly reduced in the groups of aged rats irrespective of whether they are cognitively impaired or not when compared to young rats. No difference among these groups were found for the basal values of S or P latency. 3.2. Paired pulses Paired pulses stimulation with 30 ms interval resulted in an almost complete abolition of the P in the second potential ŽFig. 2A.. P2rP1 laid between 0 to 10% in all groups and no significant differences were statiscally demonstrated among them.
Fig. 1. Schematic representation of one evoked potential in the dentate gyrus after stimulation of the perforant path. Positivity upwards. A: stimulus artefact, S: slope of the f-EPSP, P: amplitude of the population spike. The P latency was measured from the beginning of the stimulus artefact to the peak of the population spike.
3.3. LTP is impaired in aged cognitiÕely impaired animals Aged cognitively impaired rats showed an impaired synaptic potentiation in all the variables measured ŽFig. 2BFig. 2C..
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3.5. NGF treatment to old cognitiÕely impaired rats improÕes LTP The alterations in LTP induction and maintenance evidenced by the old cognitively impaired rats are reproduced almost exactly by the Cit C treated animals in magnitude and time course. No significant difference could be statistically demonstrated between these two groups in any of the measured variables at any time point. In contrast, the administration of NGF to similarly impaired rats, produces an important alleviation of those deficits in synaptic plasticity. The potentiation of the f-EPSP ŽFig. 4A. in the NGF treated rats was significantly higher than that of the Cit C group, where it was completely abolished. This S potentiation in the NGF group is similar in magnitude and time course to the one showed by aged non-impaired rats. No significant differences were found between this two groups,
Fig. 2. ŽA. Paired pulse recordings with 30 ms stimulus interval showed strong inhibitory influences on the second potential. An example from a young control animal is shown. Notice the complete abolishment of the population spike on the second potential. ŽB. Long-term potentiation in a young animal. The potential above was obtained before LTP induction. Below a record from the same animal 2 min after tetanization. Notice the increase in P amplitude and the reduction in its latency. The slope of the f-EPSP was also increased. ŽC. An example of potentiation in an old impaired rat. Above: before tetanization, below: 2 min after tetanization. Notice the absence of change in the P amplitude and the slight reduction of its latency. The f-EPSP slope remained unchanged.
The potentiation of the f-EPSP was completely abolished in the cognitively impaired old rats ŽFig. 3A.. Significant differences to the young animals could be proven at every time point. The aged non-impaired rats in contrast showed an initial enhancement of S which do not differ from the young animals though it decline slowly with time, being significantly lower than young controls at 60 min. The potentiation of the population spike was delayed in old impaired rats. The P amplitude showed no increase immediately after tetanization but it increases steadily with time ŽFig. 3B.. Significant differences to the young controls were found up to 30 min. The non impaired rats showed a potentiation similar to that of the young controls. Similarly, no reduction of the P latency was initially seen in the impaired rats ŽFig. 3C., differing significantly from the young animals at all time points except at 60 min. The non impaired old rats behaved between both groups but they do show an initial potentiation of P latency. 3.4. Paired pulses Sixty minutes after LTP induction the amplitude of P1 and P2 were both increased but the inhibitory relationship between them was preserved in quality and magnitude in all groups.
Fig. 3. ŽA. Potentiation of the f-EPSP slope ŽS. expressed as percent of the basal values Ž% S.. No potentiation occurred among the impaired animals. The S potentiation among the non-impaired aged rats do not differ from the young controls up to the 60 min recording. ) indicate significant differences with respect to the young controls Ž p- 0.05, Mann–Whitney’s U test.. ŽB. Potentiation of the population spike amplitude expressed as percent Ž%P.of the basal pretetanization values. A delayed, slowly developing potentiation appears in the group of cognitively impaired aged rats. The aged, non-impaired rats do not differ from the young controls at any time point. Legends and statistics as in A. ŽC.. Potentiation of the population spike latency expressed as percent of the basal values Ž%L.. The aged, memory deficient rats showed no appreciable reduction up to 60 min corresponding to the delayed development of the P amplitude potentiation. Non-impaired animals showed a reduction which resulted slightly inferior to that of the young animals at 5 and 15 min after tetanization. Legends and statistics as in A.
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but both resulted significantly smaller than that of the young rats at 60 min after LTP induction. The potentiation of P amplitude showed also significant differences between the NGF and Cit C groups at every time point evaluated ŽFig. 4B.. A clear initial increase of P amplitude was evident in this group in contrast with the initially absent P potentiation of the Cit C group. No significant differences occurred between the NGF treated and aged non-impaired rats or the young controls regarding this variable. Significant differences between NGF and Cit C rats were also obtained with respect to the reduction of P latency which resulted more intense in the NGF treated animals ŽFig. 4C.. The magnitude and time course of this latency reduction is similar to that obtained among the young and the aged non-impaired rats. 3.6. The amelioratiÕe effects of NGF on LTP seem to be better after a longer time The NGF treated rats received the infusion for two different periods and were evaluated with different delays after the end of treatment forming thus four different Fig. 5. Potentiation of the f-EPSP slope Ž%S. Ža. 2 min after LTP induction in the four NGF groups compared with the Cit C controls. All four groups of NGF treated animals differed significantly from the Cit C group Ž ) p- 0.05, Mann–Whitney’s U test., while no difference appeared among them. Žb. 60 min after LTP induction similar results were obtained.
Fig. 4. ŽA. The potentiation of the f-EPSP slope in the NGF treated rats expressed as percent of the basal values Ž%S. was significantly greater than that of the Cit C treated controls at every time point Ž ) p- 0.05, Mann–Whitney’s U test.. Cit C rats do not differ at any time point from the aged, cognitively impaired rats Žsee Fig. 3A.. ŽB.. The potentiation of population spike amplitude in the NGF treated rats as percent Ž%P. of the basal values was also significantly increased with respect to the Cit C treated animals. Legends and statistics as in A. ŽC. The reduction in P latency after tetanization was improved in the NGF treated rats. Significant differences were also demonstrated between NGF and Cit C treated old rats at intermediate time points. Legends and statistics as in A.
groups. No significant differences were found among those groups in any variable or time measured Žsee Fig. 5.. However, there are some evidences of a better synaptic plasticity with the 3 months delay between the end of treatment and evaluation. A two ways ANOVA considering the influence of treatment duration or delay on the LTP improvement showed significant Žp - 0.05. differences only for the delay concerning the P potentiation, both in its amplitude and latency at time 2 and 5 min. Another example is shown in Fig. 6. As can be seen, 30 min after LTP induction only the two groups with 3 months delay are still significantly above the Cit C treated rats in their P amplitude potentiation. In summary, the aged rats which showed cognitive deficits in the behavioural test showed also an affected ability to potentiate the synaptic efficacy of the PP-DG synapses after tetanic stimulation as evidenced by the absent increase of the f-EPSP and the delayed potentiation of the population spike, both in its amplitude and latency. The non impaired old rats behaved much like the young controls though some differences seem to exist specially in their capacity to maintain the f-EPSP potentiation for long time. The administration of NGF to aged cognitively impaired rats improves their capacity to develop a long-term synaptic enhancement, very similar to that shown by aged
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Fig. 6. Potentiation of the P amplitude Ž%P.: Ža. 2 min after LTP induction in the four NGF groups compared with the Cit C group. All NGF groups showed significantly higher P potentiation than Cit C Ž ) p- 0.05, Mann–Whitney’s U test.. Though no differences existed among NGF groups notice that the 3 months groups showed the higher values. Žb. 30 min after LTP induction only the 3 months groups were still significantly above the Cit C group.
rats with no cognitive impairment. Slightly better results were obtained after 3 months.
4. Discussion 4.1. The impaired potentiation in aged, cognitiÕely impaired rats might be related to the reduced learning ability Learning deficits are associated with the aging process not only in humans, but also in the rat w60x. Our results indicate that aged cognitively impaired rats also show an impaired potentiation. Previous reports have demonstrated the existence of a relationship between memory deficits and different forms of synaptic plasticity in aged rats w39x including LTP in the DG w5,17,25x, CA 1 hippocampal region w18,19x, and the cerebral cortex w7x. Current opinion favours the idea that LTP-like processes are functionally related to memory w37,42,45x. Aged rats show a reduced membrane bound PKC activity w23x and adenylate cyclase w4x, as well as a reduced expression of GAP-43 protein w14x and adhesion molecules of the NCAM family w24x which might be causally related to an impaired LTP w49x. We have found an absence of f-EPSP potentiation among aged cognitively impaired rats. Previous reports
from our group and others have demonstrated that young rats bearing a lesion of the fimbria-fornix ŽFF. system can produce a short lasting Žabout 10 min. S potentiation which rapidly declines to basal levels w9,10,12x. The FF lesion provokes a subcortical deafferentation in the hippocampus and the dentate gyrus including the cholinergic fibers of septal origin, associated with a reduction of cognitive capabilities w3,29x. A decline in the number of cholinergic neurons in the basal forebrain w21,55x probably associated to a reduced basal ACh release w13x have been described in aged rats. The reduction in hippocampal cholinergic activity is accompanied by a severe impairment of the cognitive abilities of the animals, a finding well corroborated by coincident results using different paradigms of septal cholinergic dysfunction w35,40,48x. The relevance of cholinergic activity for synaptic plasticity in the dentate gyrus have been further stressed by the facilitatory effect of the coactivation of septal input on field potentials w53x and LTP w52x evoked by perforant path stimulation. The treatment with AF64A, another selective cholinotoxin, leads to an impaired f-EPSP potentiation in the CA3 hippocampal subfield w41x. One can hypothesize that the impaired S potentiation showed by the aged, cognitively-impaired rats is attributable to a reduced cholinergic activity in the hippocampus. This line of reasoning is reinforced by the finding of a reduced choline acetyltransferase ŽChAT. activity in the hippocampal formation of an identical group of aged-impaired rats in our laboratory wFernandez et al., in ´ preparationx. The delayed P potentiation, however, might have a different origin. In fact, our results with FF-lesioned rats showed no impairment of P potentiation w9,10x, which is coincident with the reported non-essential role of the cholinergic system on P potentiation among AF64A lesioned rats w2x. An age-associated loss of granule cells and synaptic contacts in the dentate gyrus wreviewed in w25xx as well as a reduction in glutamate neurotransmission to the hippocampus have been found in aged rats w43x which might be related to reduced learning abilities and amplitude of the evoked potentials w26,33,46x. LTP is known to depend on the activation of AMPA, NMDA and metabotropic glutamate receptors w44,49,51x. A reduced glutamatergic transmission might affect the potentiation of both, the population spike and the f-EPSP. This would explain the absence in aged-impaired animals of the short-lasting initial S potentiation observed in FF-lesioned rats w9,10,12x. The delayed P potentiation might correspond the so-called voltage-dependent-calcium-channels-LTP ŽVDCC-LTP. w58x considering that an increased Ica through voltage activated channels have been demonstrated in hippocampal neurons af aged rats w59x. It must be mentioned that ketamine anaesthesia involves a reversible blockade of NMDA receptors w31x. We have attempt to reduce this effect by using ketamine only to induce the initial narcose. As the recordings were started about two
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hours after the injection of ketamine, we believe that a blockade of NMDA receptors would be very low, if still present. In other case, a differential sensitivity or affinity to ketamine among aged-impaired rats should be supposed because such effect was not observed in the young controls or the aged non-impaired rats. Paired pulses inhibition is based on the activity of local circuits of retroactive inhibition w50x. This short-term form of synaptic plasticity is not altered by the aging process neither in basal conditions nor after LTP induction as our results seem to indicate. The activity of this circuits remains unchanged after LTP induction also in young animals w16x for the medial portion of the PP. Therefore the impaired LTP observed in aged rats might not be attributed to a differential activity of this local circuits in memory deficient rats. 4.2. NGF treatment to aged cognitiÕely impaired rats restores LTP NGF prevents the degeneration and promotes the maintenance of a cholinergic phenotype of septal neurons after lesion of their axons or in aged rats and primates w20,27,36,63x which correlates with an improvement of memory w22,62x. It is tempting to speculate that the age related cholinergic deficit is caused by a reduced production of NGF in the hippocampus. Though normal levels of NGF have been reported in aged rats w28x the recent demonstration of cholinergic hypofunction and spatial learning impairments after NGF deprivation in young adult rats w61x opens new expectancies on this topic. Our results show that chronic intraventricular infusion with NGF leads to an improvement of the synaptic plastic capabilities of the treated animals, exhibiting a potentiation almost identical to that shown by non-impaired animals. Trophic factors like BDNF, NT-3, EGF and others can exert a direct facilitatory influence on LTP w1,32,34,54x but NGF seems to be deprived of such action w32,34x and can even have a direct antagonist action on LTP expression in acute experiments w56x. According to this, and to the time elapsed between the end of infusion and the electrophysiological studies is hard to conceive that the beneficial effects of NGF reported in the present paper can be attributed to a direct action of NGF. We believe instead, that the observed NGF effects on LTP are indirect and mediated by its trophic influences on cholinergic function, and possibly other afferent systems. According to the previously analysed deficits in LTP showed by cognitive-impaired old rats, an enhanced cholinergic activity in the hippocampus might explain the improved S potentiation. Evidences from our group wFernandez, in preparationx using a similar paradigm shown a recovery of ChAT activity in the hippocampus after NGF. Grafting of cholinergic fetal neurons to the hippocampus of aged or FF-lesioned rats improve their
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mnemonic capabilities wsee w57x for a reviewx, and restore the impaired S potentiation w10x. As previously discussed, there are reasons to believe that factors other than cholinergic denervation might be involved in the deficient P potentiation. The demonstration of receptors to NGF of the trkA type in the hippocampus itself w15x and of protective actions of NGF on hippocampal neurons w64x, opens new possibilities of explaining the effects of NGF on LTP and memory based on autocrine or paracrine local influences. Neurotrophic influences are exerted through cellular processes such as gene activation, neurite growth, sprouting of axon collaterals, among others, leading to a modified cell activity and a remodeling of its synapses. Those processes require time to be fully expressed, a fact that can be in correspondence with the slightly better results obtained after three months of the NGF treatment. The duration of treatment Ž14 or 28 days. seemed not to play an essential role provided an adequate dose for a minimal time is given. This conclusion is reinforced by the results obtained by our group in identically treated rats showing that ChAT recovery in the hippocampus and memory improvement are better in rats evaluated 3 months than those evaluated 1 week after treatment wFernandez in ´ preparationx. 4.3. Final considerations Our results add further evidences to the reported impairment of LTP in aged cognitively impaired rats and provide the first evidence that NGF treatment improves the decreased synaptic plasticity of aged rats suffering cognitive impairments. This enhanced capability might be functionally related to the reported effect of NGF improving memory processing in aged rats. Both findings indirectly stressed the presumed functional relationship between LTP and memory. Additionally, our findings support the proposed therapeutic use of NGF to treat age-related cognitive disorders including Alzheimer’s disease. The fact that NGF does not restore LTP to the level of young animals could probably indicate that other cooperative agents should be necessary to obtain a maximal restoration. Glutamatergic agonists appear as logical candidates but metabolic nootropics, like orotic acid and its derivatives, with a demonstrated ability to improve LTP and related processes w8,38x can also be considered. The application of such therapy to humans requires, however, the solution of technical and experimental problems which should be solved in the coming future.
Acknowledgements The cooperation of Dr. Mario Fajardo and the CIREN’s Dental care Service is acknowledged with gratitude.
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Research report
Chronic intraventricular infusion with NGF improves LTP in old cognitively-impaired rats Jorge A. Bergado ) , Caridad I. Fernandez, Andres Odalys Gonzalez ´ ´ Gomez-Soria, ´ ´ International Centre for Neurological Restoration, AÕe. 25 a 15805, Cubanacan, ´ Playa 12100, Ciudad de La Habana, Cuba Accepted 30 April 1997
Abstract Aged Ž21 months. cognitively-impaired male Sprague–Dawley rats received intraventricular infusion of nerve growth factor ŽNGF. or cytochrome C ŽCit C. for 14 or 28 days using miniosmotic pumps and were evaluated either 1 week or 3 months after treatment. Groups of untreated young, aged-impaired and aged non-impaired rats were also evaluated. Under narcose recording and stimulating electrodes were stereotactically implanted in the dentate gyrus and the perforant path. The stimulation intensity was individually adjusted to obtain a half-maximal population spike ŽP. for test stimuli and a quarter-maximal for tetanization. The amplitude and latency of P and the slope ŽS. of the field EPSP were determined before and at 2, 5, 15, 30 and 60 min after tetanization at 400 Hz. Paired stimuli at 30 ms interval were also applied before and after tetanization. Aged, cognitively impaired rats showed an absent S potentiation and a delayed P potentiation, both in amplitude and latency, while non-impaired rats behaved like the young controls. Paired pulse inhibition showed no difference among groups before or after tetanization suggesting that the impaired potentiation is not due to an increased retroactive inhibition. NGF treatment ameliorates LTP deficits to levels equivalent to non-impaired rats, while Cit C controls showed no improvement. No differences appear among NGF treated groups, but evidence suggest that the animals evaluated 3 months after treatment developed a stronger potentiation. q 1997 Elsevier Science B.V. Keywords: Long-term potentiation; Aging; Nerve growth factor
1. Introduction Nerve growth factor ŽNGF. is the best characterized member of the neurotrophin family exhibiting actions on the peripheral and central nervous system. Only the population of cholinergic neurons distributed along the basal forebrain nuclei and the cholinergic striatal interneurons respond to NGF within the brain w30x. Basal forebrain cholinergic neurons project to the hippocampus and the cerebral cortex where they presumably receive trophic support from target-derived NGF. A gradual decline in the size and number of this cholinergic population with increasing age have been correlated to the progressive memory decline which accompanies aging, especially its pathological variants like Alzheimer’s disease w6,47x. This hypothesized pathological causality together with the known trophic action of NGF on cholinergic neurons provides a rationale for the experi) Corresponding author. [email protected]
Fax: q53
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332-420; E-mail:
0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 6 1 0 - 0
mental studies on the possible therapeutic usefulness of a NGF-based therapy for age-related cognitive impairments. The experimental evidence shows, coincidently, that intracerebral infusion of NGF to aged animals leads to an improvement of their mnemonic capabilities w22,28x which has been attributed to the enhanced cholinergic function in the targets, specially the hippocampus. Since its discovery in the dentate gyrus ŽDG., hippocampal long-term potentiation ŽLTP. has been proposed as a correlate of long-term memory w11x. This assumption is based mainly on analogies because no conclusive evidence linking LTP and memory has yet been presented, but beyond this unsolved issue, most theories of learning consider that Hebbian plastic modifications of synaptic connectivity occur during the formation of memory engrams. LTP is a long-lasting form of synaptic plasticity and appears, therefore, as a suitable model to study the consequences of NGF treatment on synaptic plasticity. Furthermore, LTP have been found to be altered in aged rats in direct relationship to senescent memory dysfunction w5,17x. The question emerges of whether NGF treatment
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can alleviate the deficient LTP exhibited by aged, cognitively deficient rats. The present paper constitutes the first approach to this fundamental aspect. Our results indicate that chronic intraventricular infusion of NGF to old-impaired rats is able to improve LTP, adding support to the proposed therapeutic use of NGF for the treatment of age-associated memory dysfunctions and offering new perspectives to the interpretation of behavioural results.
2. Materials and methods 2.1. Animals Ten young Ž2 months. and 66 old Ž21 months at the beginning of the experiment. Sprague–Dawley male rats obtained from CENPALAB ŽHavana. were used. The rats were maintained in plastic translucent cages Ž5 animals per cage. under controlled conditions of humidity and temperature with 12:12 h light–dark cycle and free access to food and water during the whole experiment. 2.2. BehaÕioural test Cognitive capacities were evaluated using the Morris Water Maze ŽMWM.. A circular pool with a hidden platform was used. Rats were entered the pool using a pseudorandom distribution of the starting points and the time they need to find the platform was measured. A maximal time of 60 s was allowed in each trial. If after that time the rat was unable to find the escape platform, they were gently carried upon the platform and allowed to stay there for 30 s before the next trial. Eight sessions were performed daily during 5 consecutive days. According to their results, compared with those of a group of ten young rats, old animals were classified as cognitively impaired when their mean latency to find the platform was at least 3
standard deviations above the mean latency of the young animals. The cognitively impaired rats were then randomly assigned to one of the experimental groups. From the non impaired rats some animals were selected at random to constitute one experimental group Žsee Table 1.. The rest of the non impaired rats were discarded an not considered part of the experimental protocol. 2.3. NGF treatment Groups of old-cognitively impaired rats received either 5S murine NGF obtained from mouse submaxillary gland, or Cytochrome C ŽCit C. protein treatment using 14 days continuous delivery miniosmotic pumps ŽAlzet, CA.. Pumps were filled with one of the proteins dissolved in saline and primed before being intrascapullary implanted under ketamine anaesthesia. The pumps were connected through a polyethylene catheter to a stainless steel cannula implanted into the right ventricle. Those animals receiving the substance for 28 days were reanesthetised two weeks after the initial implant and the empty pump was changed by a full one containing the same substance. The concentration of either protein in the pump was 22 m grml, equivalent to an infusion of 1.2 m grday. 2.4. Grouping According to the treatment received the rats were divided in different groups Žsee Table 1.. A control group of young rats, a group of aged non-cognitively impaired rats, and a group of aged animals with cognitive impairment received no treatment before the electrophysiological study. Meanwhile, several subgroups of aged-impaired rats received NGF or Cit C treatment for 14 or 28 days of continuous infusion and were evaluated 1 week or 3 months after the end of substance delivery. As no statistical difference was found among the Cit C treated animals when compared to each other, they were considered as one
Table 1 Experimental groups Group
n
Animals
Substance
Infusion time
Delay
Control Aged non-impaired Aged impaired C 14–1 C 14–3 C 28–1 C 28–3 Cit C N 14–1 N 14–3 N 28–1 N 28–3 NGF
10 9 7 5 5 8 7 25 5 8 6 6 25
Young rats Old rats without cognitive impairment Old rats cognitively impaired Old impaired rats Old impaired rats Old impaired rats Old impaired rats Old impaired rats Old impaired rats Old impaired rats Old impaired rats Old impaired rats Old impaired rats
– – – Cit C Cit C Cit C Cit C Cit C NGF NGF NGF NGF NGF
– – – 14 days 14 days 28 days 28 days
– – – 1 week 3 months 1 week 3 months
14 days 14 days 28 days 28 days
1 week 3 months 1 week 3 months
As no difference was found among the Cit C treated groups in any of the measured variables they were further considered as a single Cit C group Ž n s 25.. The NGF-receiving rats were also grouped in this way for the initial presentations of results Ž n s 25..
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single group ŽCit C.. For the sake of clarity in the presentation of results all animals receiving NGF were also presented as one group ŽNGF. at some instances. 2.5. Electrophysiology For the electrophysiological analysis the rats were anaesthetised using an initial bolus of ketamine clorhydrate Ž3.6 mgrkg. which provides a narcose lasting about 45–60 min, suplemented with chloral hydrate Ž140 mgrkg. every 60 min to maintain narcose during the recording time. The rats were mounted in a stereotaxic frame ŽDavid Kopf, USA.. One monopolar recording electrode ŽRNE 300, Rhodes Med. Inst.. and a bipolar stimulating electrode ŽNE 200, Rhodes Med. Inst.. were lowered at the coordinates; AP: y3.8, L: 2.0, V: 4.0 mm. from bregma aiming the hilus of the DG for recording, and AP: y7.5, L: 4.0, V: 4.5 mm. also from bregma, in the medial portion of the perforant path ŽPP. for stimulation. The vertical position of both electrodes was adjusted under inspection of the evoked potentials to obtain the best sensitivity. Two miniscrews were placed over both frontal bones to serve as earth and indifferent electrode respectively. Constant current square single pulses Ž0.1 ms. were delivered to the PP using a SEN-3301 electronic stimulator ŽNihon Kohden, Japan. connected to the animal through a SS-104J isolator ŽNihon Kohden, Japan.. The evoked potentials were registered using a VC-11 memory osciloscope ŽNihon Kohden, Japan. filtered between 1.5 Hz–3 KHz. Four consecutive responses at 0.2 Hz were averaged with a QC-111J analyser ŽNihon Kohden, Japan. and measured on-line. Three variables were determined on each recorded evoked potential Žsee Fig. 1. The slope ŽS. of field excitatory postsynaptic potential Žf-EPSP. was calculated as DVrDt on the first positive deflection of the potential. The amplitude of the population spike ŽP. and its latency from the beginning of the stimulus artifact ŽP-latency. were also measured. An inputroutput curve was constructed for each animal using the P amplitude as measure of the output. On this curve the stimulation intensity needed to evoke a half maximal P was determined and used for every test record-
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ing. For the induction of LTP an intensity evoking a quarter maximal P was utilized. Three control records with 5 min interval were made before the induction of LTP and the values of S, P amplitude and P latency measured and averaged to obtain the basal values of the three variables. Paired pulse stimulation Ž30 ms interstimulus interval. was also performed before the induction of LTP. The degree of inhibition or facilitation was determined using the amplitude ratio ŽP2rP1 . in both potentials expressed as percent. Paired pulse stimulation was also carried out after the last test recording. LTP was induced using 10 trains of 10 impulses each Ž0.1 ms square pulses at 400 Hz. with 10 s intertrain interval. The intensity was adjusted as described above. Test recordings were performed 2, 5, 15, 30 and 60 min after LTP induction. S, P amplitude, and P latency were determined in each record and expressed as percent the respective basal value. 2.6. Statistics Comparisons were performed using the non-parametric analysis of variance of Kruskall–Wallis followed by a Mann–Whitney’s U-test to asses differences between pairs of groups. In every case a p value inferior to 0.05 was considered significant.
3. Results Though all groups of animals were processed in parallel, the results of the non-treated animals will be described first for the sake of clarity. 3.1. Basal Õalues The basal P amplitude measured before the induction of LTP was significantly reduced in the groups of aged rats irrespective of whether they are cognitively impaired or not when compared to young rats. No difference among these groups were found for the basal values of S or P latency. 3.2. Paired pulses Paired pulses stimulation with 30 ms interval resulted in an almost complete abolition of the P in the second potential ŽFig. 2A.. P2rP1 laid between 0 to 10% in all groups and no significant differences were statiscally demonstrated among them.
Fig. 1. Schematic representation of one evoked potential in the dentate gyrus after stimulation of the perforant path. Positivity upwards. A: stimulus artefact, S: slope of the f-EPSP, P: amplitude of the population spike. The P latency was measured from the beginning of the stimulus artefact to the peak of the population spike.
3.3. LTP is impaired in aged cognitiÕely impaired animals Aged cognitively impaired rats showed an impaired synaptic potentiation in all the variables measured ŽFig. 2BFig. 2C..
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3.5. NGF treatment to old cognitiÕely impaired rats improÕes LTP The alterations in LTP induction and maintenance evidenced by the old cognitively impaired rats are reproduced almost exactly by the Cit C treated animals in magnitude and time course. No significant difference could be statistically demonstrated between these two groups in any of the measured variables at any time point. In contrast, the administration of NGF to similarly impaired rats, produces an important alleviation of those deficits in synaptic plasticity. The potentiation of the f-EPSP ŽFig. 4A. in the NGF treated rats was significantly higher than that of the Cit C group, where it was completely abolished. This S potentiation in the NGF group is similar in magnitude and time course to the one showed by aged non-impaired rats. No significant differences were found between this two groups,
Fig. 2. ŽA. Paired pulse recordings with 30 ms stimulus interval showed strong inhibitory influences on the second potential. An example from a young control animal is shown. Notice the complete abolishment of the population spike on the second potential. ŽB. Long-term potentiation in a young animal. The potential above was obtained before LTP induction. Below a record from the same animal 2 min after tetanization. Notice the increase in P amplitude and the reduction in its latency. The slope of the f-EPSP was also increased. ŽC. An example of potentiation in an old impaired rat. Above: before tetanization, below: 2 min after tetanization. Notice the absence of change in the P amplitude and the slight reduction of its latency. The f-EPSP slope remained unchanged.
The potentiation of the f-EPSP was completely abolished in the cognitively impaired old rats ŽFig. 3A.. Significant differences to the young animals could be proven at every time point. The aged non-impaired rats in contrast showed an initial enhancement of S which do not differ from the young animals though it decline slowly with time, being significantly lower than young controls at 60 min. The potentiation of the population spike was delayed in old impaired rats. The P amplitude showed no increase immediately after tetanization but it increases steadily with time ŽFig. 3B.. Significant differences to the young controls were found up to 30 min. The non impaired rats showed a potentiation similar to that of the young controls. Similarly, no reduction of the P latency was initially seen in the impaired rats ŽFig. 3C., differing significantly from the young animals at all time points except at 60 min. The non impaired old rats behaved between both groups but they do show an initial potentiation of P latency. 3.4. Paired pulses Sixty minutes after LTP induction the amplitude of P1 and P2 were both increased but the inhibitory relationship between them was preserved in quality and magnitude in all groups.
Fig. 3. ŽA. Potentiation of the f-EPSP slope ŽS. expressed as percent of the basal values Ž% S.. No potentiation occurred among the impaired animals. The S potentiation among the non-impaired aged rats do not differ from the young controls up to the 60 min recording. ) indicate significant differences with respect to the young controls Ž p- 0.05, Mann–Whitney’s U test.. ŽB. Potentiation of the population spike amplitude expressed as percent Ž%P.of the basal pretetanization values. A delayed, slowly developing potentiation appears in the group of cognitively impaired aged rats. The aged, non-impaired rats do not differ from the young controls at any time point. Legends and statistics as in A. ŽC.. Potentiation of the population spike latency expressed as percent of the basal values Ž%L.. The aged, memory deficient rats showed no appreciable reduction up to 60 min corresponding to the delayed development of the P amplitude potentiation. Non-impaired animals showed a reduction which resulted slightly inferior to that of the young animals at 5 and 15 min after tetanization. Legends and statistics as in A.
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but both resulted significantly smaller than that of the young rats at 60 min after LTP induction. The potentiation of P amplitude showed also significant differences between the NGF and Cit C groups at every time point evaluated ŽFig. 4B.. A clear initial increase of P amplitude was evident in this group in contrast with the initially absent P potentiation of the Cit C group. No significant differences occurred between the NGF treated and aged non-impaired rats or the young controls regarding this variable. Significant differences between NGF and Cit C rats were also obtained with respect to the reduction of P latency which resulted more intense in the NGF treated animals ŽFig. 4C.. The magnitude and time course of this latency reduction is similar to that obtained among the young and the aged non-impaired rats. 3.6. The amelioratiÕe effects of NGF on LTP seem to be better after a longer time The NGF treated rats received the infusion for two different periods and were evaluated with different delays after the end of treatment forming thus four different Fig. 5. Potentiation of the f-EPSP slope Ž%S. Ža. 2 min after LTP induction in the four NGF groups compared with the Cit C controls. All four groups of NGF treated animals differed significantly from the Cit C group Ž ) p- 0.05, Mann–Whitney’s U test., while no difference appeared among them. Žb. 60 min after LTP induction similar results were obtained.
Fig. 4. ŽA. The potentiation of the f-EPSP slope in the NGF treated rats expressed as percent of the basal values Ž%S. was significantly greater than that of the Cit C treated controls at every time point Ž ) p- 0.05, Mann–Whitney’s U test.. Cit C rats do not differ at any time point from the aged, cognitively impaired rats Žsee Fig. 3A.. ŽB.. The potentiation of population spike amplitude in the NGF treated rats as percent Ž%P. of the basal values was also significantly increased with respect to the Cit C treated animals. Legends and statistics as in A. ŽC. The reduction in P latency after tetanization was improved in the NGF treated rats. Significant differences were also demonstrated between NGF and Cit C treated old rats at intermediate time points. Legends and statistics as in A.
groups. No significant differences were found among those groups in any variable or time measured Žsee Fig. 5.. However, there are some evidences of a better synaptic plasticity with the 3 months delay between the end of treatment and evaluation. A two ways ANOVA considering the influence of treatment duration or delay on the LTP improvement showed significant Žp - 0.05. differences only for the delay concerning the P potentiation, both in its amplitude and latency at time 2 and 5 min. Another example is shown in Fig. 6. As can be seen, 30 min after LTP induction only the two groups with 3 months delay are still significantly above the Cit C treated rats in their P amplitude potentiation. In summary, the aged rats which showed cognitive deficits in the behavioural test showed also an affected ability to potentiate the synaptic efficacy of the PP-DG synapses after tetanic stimulation as evidenced by the absent increase of the f-EPSP and the delayed potentiation of the population spike, both in its amplitude and latency. The non impaired old rats behaved much like the young controls though some differences seem to exist specially in their capacity to maintain the f-EPSP potentiation for long time. The administration of NGF to aged cognitively impaired rats improves their capacity to develop a long-term synaptic enhancement, very similar to that shown by aged
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Fig. 6. Potentiation of the P amplitude Ž%P.: Ža. 2 min after LTP induction in the four NGF groups compared with the Cit C group. All NGF groups showed significantly higher P potentiation than Cit C Ž ) p- 0.05, Mann–Whitney’s U test.. Though no differences existed among NGF groups notice that the 3 months groups showed the higher values. Žb. 30 min after LTP induction only the 3 months groups were still significantly above the Cit C group.
rats with no cognitive impairment. Slightly better results were obtained after 3 months.
4. Discussion 4.1. The impaired potentiation in aged, cognitiÕely impaired rats might be related to the reduced learning ability Learning deficits are associated with the aging process not only in humans, but also in the rat w60x. Our results indicate that aged cognitively impaired rats also show an impaired potentiation. Previous reports have demonstrated the existence of a relationship between memory deficits and different forms of synaptic plasticity in aged rats w39x including LTP in the DG w5,17,25x, CA 1 hippocampal region w18,19x, and the cerebral cortex w7x. Current opinion favours the idea that LTP-like processes are functionally related to memory w37,42,45x. Aged rats show a reduced membrane bound PKC activity w23x and adenylate cyclase w4x, as well as a reduced expression of GAP-43 protein w14x and adhesion molecules of the NCAM family w24x which might be causally related to an impaired LTP w49x. We have found an absence of f-EPSP potentiation among aged cognitively impaired rats. Previous reports
from our group and others have demonstrated that young rats bearing a lesion of the fimbria-fornix ŽFF. system can produce a short lasting Žabout 10 min. S potentiation which rapidly declines to basal levels w9,10,12x. The FF lesion provokes a subcortical deafferentation in the hippocampus and the dentate gyrus including the cholinergic fibers of septal origin, associated with a reduction of cognitive capabilities w3,29x. A decline in the number of cholinergic neurons in the basal forebrain w21,55x probably associated to a reduced basal ACh release w13x have been described in aged rats. The reduction in hippocampal cholinergic activity is accompanied by a severe impairment of the cognitive abilities of the animals, a finding well corroborated by coincident results using different paradigms of septal cholinergic dysfunction w35,40,48x. The relevance of cholinergic activity for synaptic plasticity in the dentate gyrus have been further stressed by the facilitatory effect of the coactivation of septal input on field potentials w53x and LTP w52x evoked by perforant path stimulation. The treatment with AF64A, another selective cholinotoxin, leads to an impaired f-EPSP potentiation in the CA3 hippocampal subfield w41x. One can hypothesize that the impaired S potentiation showed by the aged, cognitively-impaired rats is attributable to a reduced cholinergic activity in the hippocampus. This line of reasoning is reinforced by the finding of a reduced choline acetyltransferase ŽChAT. activity in the hippocampal formation of an identical group of aged-impaired rats in our laboratory wFernandez et al., in ´ preparationx. The delayed P potentiation, however, might have a different origin. In fact, our results with FF-lesioned rats showed no impairment of P potentiation w9,10x, which is coincident with the reported non-essential role of the cholinergic system on P potentiation among AF64A lesioned rats w2x. An age-associated loss of granule cells and synaptic contacts in the dentate gyrus wreviewed in w25xx as well as a reduction in glutamate neurotransmission to the hippocampus have been found in aged rats w43x which might be related to reduced learning abilities and amplitude of the evoked potentials w26,33,46x. LTP is known to depend on the activation of AMPA, NMDA and metabotropic glutamate receptors w44,49,51x. A reduced glutamatergic transmission might affect the potentiation of both, the population spike and the f-EPSP. This would explain the absence in aged-impaired animals of the short-lasting initial S potentiation observed in FF-lesioned rats w9,10,12x. The delayed P potentiation might correspond the so-called voltage-dependent-calcium-channels-LTP ŽVDCC-LTP. w58x considering that an increased Ica through voltage activated channels have been demonstrated in hippocampal neurons af aged rats w59x. It must be mentioned that ketamine anaesthesia involves a reversible blockade of NMDA receptors w31x. We have attempt to reduce this effect by using ketamine only to induce the initial narcose. As the recordings were started about two
J.A. Bergado et al.r Brain Research 770 (1997) 1–9
hours after the injection of ketamine, we believe that a blockade of NMDA receptors would be very low, if still present. In other case, a differential sensitivity or affinity to ketamine among aged-impaired rats should be supposed because such effect was not observed in the young controls or the aged non-impaired rats. Paired pulses inhibition is based on the activity of local circuits of retroactive inhibition w50x. This short-term form of synaptic plasticity is not altered by the aging process neither in basal conditions nor after LTP induction as our results seem to indicate. The activity of this circuits remains unchanged after LTP induction also in young animals w16x for the medial portion of the PP. Therefore the impaired LTP observed in aged rats might not be attributed to a differential activity of this local circuits in memory deficient rats. 4.2. NGF treatment to aged cognitiÕely impaired rats restores LTP NGF prevents the degeneration and promotes the maintenance of a cholinergic phenotype of septal neurons after lesion of their axons or in aged rats and primates w20,27,36,63x which correlates with an improvement of memory w22,62x. It is tempting to speculate that the age related cholinergic deficit is caused by a reduced production of NGF in the hippocampus. Though normal levels of NGF have been reported in aged rats w28x the recent demonstration of cholinergic hypofunction and spatial learning impairments after NGF deprivation in young adult rats w61x opens new expectancies on this topic. Our results show that chronic intraventricular infusion with NGF leads to an improvement of the synaptic plastic capabilities of the treated animals, exhibiting a potentiation almost identical to that shown by non-impaired animals. Trophic factors like BDNF, NT-3, EGF and others can exert a direct facilitatory influence on LTP w1,32,34,54x but NGF seems to be deprived of such action w32,34x and can even have a direct antagonist action on LTP expression in acute experiments w56x. According to this, and to the time elapsed between the end of infusion and the electrophysiological studies is hard to conceive that the beneficial effects of NGF reported in the present paper can be attributed to a direct action of NGF. We believe instead, that the observed NGF effects on LTP are indirect and mediated by its trophic influences on cholinergic function, and possibly other afferent systems. According to the previously analysed deficits in LTP showed by cognitive-impaired old rats, an enhanced cholinergic activity in the hippocampus might explain the improved S potentiation. Evidences from our group wFernandez, in preparationx using a similar paradigm shown a recovery of ChAT activity in the hippocampus after NGF. Grafting of cholinergic fetal neurons to the hippocampus of aged or FF-lesioned rats improve their
7
mnemonic capabilities wsee w57x for a reviewx, and restore the impaired S potentiation w10x. As previously discussed, there are reasons to believe that factors other than cholinergic denervation might be involved in the deficient P potentiation. The demonstration of receptors to NGF of the trkA type in the hippocampus itself w15x and of protective actions of NGF on hippocampal neurons w64x, opens new possibilities of explaining the effects of NGF on LTP and memory based on autocrine or paracrine local influences. Neurotrophic influences are exerted through cellular processes such as gene activation, neurite growth, sprouting of axon collaterals, among others, leading to a modified cell activity and a remodeling of its synapses. Those processes require time to be fully expressed, a fact that can be in correspondence with the slightly better results obtained after three months of the NGF treatment. The duration of treatment Ž14 or 28 days. seemed not to play an essential role provided an adequate dose for a minimal time is given. This conclusion is reinforced by the results obtained by our group in identically treated rats showing that ChAT recovery in the hippocampus and memory improvement are better in rats evaluated 3 months than those evaluated 1 week after treatment wFernandez in ´ preparationx. 4.3. Final considerations Our results add further evidences to the reported impairment of LTP in aged cognitively impaired rats and provide the first evidence that NGF treatment improves the decreased synaptic plasticity of aged rats suffering cognitive impairments. This enhanced capability might be functionally related to the reported effect of NGF improving memory processing in aged rats. Both findings indirectly stressed the presumed functional relationship between LTP and memory. Additionally, our findings support the proposed therapeutic use of NGF to treat age-related cognitive disorders including Alzheimer’s disease. The fact that NGF does not restore LTP to the level of young animals could probably indicate that other cooperative agents should be necessary to obtain a maximal restoration. Glutamatergic agonists appear as logical candidates but metabolic nootropics, like orotic acid and its derivatives, with a demonstrated ability to improve LTP and related processes w8,38x can also be considered. The application of such therapy to humans requires, however, the solution of technical and experimental problems which should be solved in the coming future.
Acknowledgements The cooperation of Dr. Mario Fajardo and the CIREN’s Dental care Service is acknowledged with gratitude.
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